This invention relates to a method for the removal of a nitrogen containing heterocyclic or aromatic compound, comprising at least one nitro group. The method of the invention consists of converting at least one nitro group into an amine group by means of a reducing agent. The reducing agent can be a micro-organism capable of reducing said, at least one nitro group into an amine group, or can be hydrogen in the presence of a Pd/C catalyst.
This invention also relates to a method for the synthesis of 5-amino-1,2,4-triazole-3-one from 5-nitro-1,2,4-triazole-3-one by said method of conversion.
This invention also relates to a colony of the type Bacillus licheniformis capable of converting one or more nitro groups of nitrogen containing heterocyclic compounds into one or more amine groups.
The nitrogen containing heterocyclic compound can be, for example, a triazine or a triazole. These compounds constitute a heterogeneous family of nitrogen containing molecules several of which are used in industry and in agriculture. Although the toxicity of these compounds has not always been given prominence, they can nevertheless constitute a source of pollution, when released into the natural environment.
An example of such a compound is ONTA or 1,2-dihydro-5-nitro-1,2,4-triazole-3-one. This compound is explosive and it can reach concentrations of about 15 g/l in certain solutions such as industrial processing effluents stemming from the manufacture of explosives. These effluents are pollutants according to the legislation currently in force and are at present being stored in this state while awaiting a suitable treatment process.
The method of the invention enables precisely such solutions to be treated, solutions comprising, in particular, a compound such as ONTA, that is to say an explosive nitrogen containing heterocyclic compound, comprising at least one nitro group, by converting this explosive nitrogen containing heterocyclic compound into a non-explosive compound.
In the example mentioned above, the method of the invention allows one to convert the nitro group of the ONTA into an amine group to form 5-amino-1,2,4-triazole-3-one.
Furthermore, the 5-amino-1,2,4-triazole-3-one formed is a compound of value, since it can be a precursor for the synthesis of compounds, having, for example antiviral activity such as ribavirine, and pirazomicine, an inhibiting activity for the NO-synthases (NO.S.) such as 3-amino-1,2,4-triazine and aminoguanidine, a herbicide activity such as 5-amino-1,2,4-triazole, an anti-parasitic activity and a cytotoxic activity.
The method of the invention therefore makes it possible, in certain cases to convert a compound of no value into a compound of value.
Another aspect of the method of the invention is the synthesis of the heterocyclic amine 5-amino-1,2,4-triazole-3-one from 5-nitro-1,2,4-triazole-3-one.
The aromatic compound comprising at least one nitro group can be, for example, a benzene ring with one or more nitro groups, for example a compound chosen from the group comprising 2,6-dinitroaniline, 1,3-dinitrobenzene, 2-nitroaniline and ortho-, meta- and para-nitrobenzoic acids.
The aromatic amines and nitrogen containing heterocyclic amines are often obtained by reduction of the nitrated analogue. This reduction is catalyzed by various methods using reducing agents such as zinc, iron or tin, in an acid medium or hydrides such as lithium aluminum hydride, or catalytic hydrogenation. However, when the aromatic amines are complex, that is to say when they include sensitive functional groups apart from the nitro group, numerous secondary reactions can occur on these groups that then reduces the yield of the synthesis of the targeted aromatic amine.
For example, the reduction of ONTA by catalytic hydrogenation in the presence of platinum oxide can affect the aromaticity of the molecule in addition to the nitro group.
Furthermore, the experimental conditions for these synthesis methods are often drastic and dangerous.
This invention relates to a method for the at least partial removal of at least one nitrogen containing heterocyclic compound or aromatic compound comprising at least one nitro group, present in an effluent, said method comprising the following steps:
bringing the solution of the nitrogen containing heterocyclic compound or aromatic compound to be removed into contact with a reducing agent in order to convert said nitro group or groups of the heterocyclic compound or aromatic compound into one or more amine groups, and
separation of the reducing agent from the solution of the nitrogen containing heterocyclic compound or aromatic compound, at least a part of whose nitro groups has been converted into amine groups.
According to the method of the invention, the solution of the nitrogen containing heterocyclic compound or aromatic compound comprising at least one nitro group can contain a single compound or several different compounds. This solution can be an industrial processing effluent arising from the manufacture of explosives, an industrial effluent, foul water and more generally a solution comprising nitrogen containing heterocyclic compounds or aromatic compounds comprising at least one nitro group that it is necessary to treat by converting one or more nitro groups into amine groups. This solution will also be referred to in what follows as the solution to be treated, and the nitrogen containing heterocyclic compound or aromatic compound comprising at least one nitro group will also be referred to in what follows, respectively as a nitrogen containing heterocyclic compound to be treated or an aromatic compound to be treated.
According to the method of the invention, the reducing agent can be hydrogen in the presence of a Pd/C catalyst.
The hydrogenation catalyst Pd/C, or palladium on carbon, can be used at a quantity of from 5 to 15% by weight with respect to the quantity of nitrogen containing heterocyclic compound or aromatic compound to be removed, preferably at a quantity of 10% by weight. The effluent can, for example, be an aqueous solution or methanol.
According to the method of the invention, the reducing agent can be a micro-organism.
According to the method of the invention, the nitrogen containing heterocyclic compound comprising at least one nitro group can be a triazole.
According to the method of the invention, when the nitrogen containing heterocyclic compound to be treated is a triazole, it can be a compound with the following formula (I): 
in which the R1 and R2 groups, which can be identical or different, represent H, or a substituent group chosen from within the group comprising xe2x80x94NO2, xe2x80x94OH, xe2x80x94COOH, xe2x80x94Cl, xe2x80x94NH2, or a cyclic or linear glucose unit, non-substituted or substituted by one or more groups chosen independently from within the group comprising for example an acetyl or a tolyl group or a linear or cyclic alkyl group, comprising from 1 to 10 atoms of carbon, non-substituted or substituted by one or more groups chosen independently from within the group comprising xe2x80x94NO2, xe2x80x94OH, xe2x80x94COOH, xe2x80x94Cl, xe2x80x94NH2. For example, when R1 and/or R2 is a glucose unit, it can be ribose, deoxyribose, etc non-substituted or substituted.
According to the method of the invention, the compound of formula (I) can be converted into a compound with the following formula (II): 
in which the groups R3 and R4, which can be identical or different, represent H, a substituent group chosen from within the group comprising xe2x80x94NO2, xe2x80x94OH, xe2x80x94COOH, xe2x80x94Cl, xe2x80x94NH2, or a cyclic or linear glucose unit, non-substituted or substituted by one or more groups chosen independently from within the group comprising for example an acetyl or a tolyl group or a linear or cyclic alkyl group, with 1 to 10 atoms of carbon, non-substituted or substituted by one or more groups chosen independently from among xe2x80x94NO2, xe2x80x94OH, xe2x80x94COOH, xe2x80x94Cl, xe2x80x94NH2. For example, when R1 and/or R2 is a glucose unit, it can be ribose, deoxyribose, xcex2-D-ribofuranosyl, etc.
When R1 is a xe2x80x94NO2 group, this group can be reduced to the corresponding amine and R3 can then be an xe2x80x94NH2 group. Similarly, when R2 is a xe2x80x94NO2 group, this group can be reduced to the corresponding amine and R4 can then be an xe2x80x94NH2 group.
The aromatic compound comprising at least one nitro group to be treated can be for example, a mono-, di- or tri-nitrobenzene, 2-nitroaniline and ortho-, meta- and para-nitrobenzoic acids.
For example, when 2,6-dinitroaniline is treated, one can obtain 2-amino-6-nitroaniline, when 1,3-dinitrobenzene is treated, one can obtain 1-amino-3-nitrobenzene, when 2-nitroaniline is treated, one can obtain 2-aminoaniline and when the ortho-, meta- and para-nitrobenzoic acids, one can obtain ortho-, meta- and para-aminobenzoic acids respectively.
According to the invention, the micro-organism capable of converting said nitro group or groups into an amino group or groups can be selected by the screening of micro-organisms that are living or which could live, in an industrial processing effluent arising from the manufacture of explosives, in foul water, and more generally in a medium, referred to below as the source medium, comprising nitrogen containing heterocyclic compounds comprising preferably at least one nitro group. For example, the screening can be carried out starting with a sample that can be treated according to the method of the invention, for example a sample from an industrial processing effluent arising from the manufacture of explosives, or from purification water that includes organic compounds that comprise nitro groups, nitrated aromatic compounds, nitrogen containing heterocyclic compounds etc.
According to the method of the invention, the micro-organism capable of converting said nitro group or groups into an amine group or groups can be selected from a laboratory collection.
According to the method of the invention, the selection by screening can be carried out, for example, from a culture of micro-organisms living in a source medium such as those mentioned above. This culture is preferably carried out on a rich culture medium, in a way that enables life to be maintained and that enables one to cultivate a maximum of the micro-organisms present in this source medium. Moreover, the culture medium is preferably solid so that the micro-organisms can form colonies.
An example of a rich culture medium for the selection by screening is a medium that comprises for 1 liter of water:
The medium is sterilized at 120xc2x0 C. for 20 minutes before use.
When the number of micro-organisms is too great in the source medium, the culture of these micro-organisms on a rich solid culture medium will be preceded by a dilution of a sample of the source medium so as to be able to form isolated colonies of these micro-organisms.
An analysis of the capacity of each of the colonies formed on the rich solid medium to reduce nitro groups of nitrogen containing heterocyclic compounds to be treated and an analysis of the biochemical nature of each of these colonies respectively enables one to give prominence to and to identify the micro-organisms that can be used in accordance with the method of the invention.
Preferably, according to the invention, the micro-organism is a micro-organism that is widespread in the natural environment and does not manifest any particular toxicity.
According to the invention, the micro-organism, in addition to converting one or more nitro groups of nitrogen containing heterocyclic compounds or aromatic compounds into an amine group, can metabolize the nitrogen containing heterocyclic compound or aromatic compound comprising said amine group or groups in such a way that this compound is partially or totally degraded.
The micro-organism can, for example, be one or more filamentous fungi and/or one or more bacteria.
When the micro-organism is a bacterium, it may be of the Bacillus licheniformis type.
The preferred Bacillus licheniformis can be the Bacillus licheniformis colony LCM2 filed with the Collection Nationale de Cultures de Microoganismes (CNCM), Institut Pasteur, located at 25, Rue du Docteur Roux, F-75724 Paris Cedex 15, France. The deposit was filed Nov. 18, 1997 under the accession number I-1915 for the deposit.
According to the method of the invention, the micro-organism Bacillus licheniformis is, for example, suitable for the conversion of the nitrogen containing heterocyclic compound to be treated with the following formula (III): 
into a nitrogen containing heterocyclic compound with the following formula (IV): 
According to the method of the invention, bringing the solution of said nitrogen containing heterocyclic compound to be treated into contact with a micro-organism capable of reducing said nitro group or groups into amine groups is carried out by mixing a biomass of said micro-organism with said solution.
The biomass of the micro-organism can be obtained through a culture of the one or more micro-organisms selected by screening, in a rich, solid or liquid culture medium, preferably a liquid culture medium.
A rich medium that can be used to develop the biomass, when the micro-organism is Bacillus licheniformis is a medium comprising, for a liter of water:
The medium is sterilized at 120xc2x0 C. for 20 minutes before use.
When a sufficient quantity of biomass is obtained, and when the culture medium is a liquid, this biomass can be recovered by compacting it by centrifugation of the culture medium.
According to the method of the invention, the biomass can be obtained from one or more types of micro-organisms capable of reducing one or more nitro groups into amine groups in accordance with the method of the invention.
The biomass mixed with the solution of nitrogen containing heterocyclic compound to be treated for the bringing into contact must be in a sufficient quantity to allow the conversion of the one or more nitro groups of the nitrogen containing heterocyclic compounds or aromatic compounds to be treated which are present in this solution. The quantity of biomass to be used can be determined, for example, as a function of the quantity of compounds that comprise nitro groups to be converted into amine groups. This quantity of biomass may also be determined by means of tests in which the quantity for conversion of nitro groups of a nitrogen containing heterocyclic compound present in a solution is measured in relation to the quantity of biomass of micro-organism mixed with this solution, the concentration of nitrogen containing heterocyclic compounds to be treated, the duration of the tests being identical for each test.
The biomass can be mixed with the solution of nitrogen containing heterocyclic compound to be treated by means of a traditional stirrer, for example a rotary stirrer, at a stirring speed that can range from 50 to 250 rpm, for example 100 rpm.
The stirring can be maintained for the entire duration of the contact between the biomass of a micro-organism and the nitrogen containing heterocyclic compound to be treated in a manner that optimizes this bringing into contact. The mixture of biomass and solution of the nitrogen containing heterocyclic compound to be treated is also referred to below as the incubation mixture.
Preferably, according to the method of the invention, the solution of the nitrogen containing heterocyclic compound comprising at least one nitro group, or the incubation mixture, can be neutralized to a pH value that permits the conversion of the one or more nitro groups into amine groups.
A series of tests consisting of measuring the quantity of compounds comprising nitro groups converted in an incubation mixture as a function of the pH can enable one to determine a pH range that permits this conversion, that is to say, favors this conversion.
For example, when the micro-organism is Bacillus licheniformis, the pH of the solution of the nitrogen containing heterocyclic compound comprising at least one nitro group, and/or the incubation mixture, is neutralized to a value within the pH range of 5.5 to 8 for bringing it into contact with the micro-organism.
Moreover, when the nitrogen containing heterocyclic compound to be treated is ONTA, the amine formed is stable and is not subject to any subsequent transformation while the pH of the incubation mixture is maintained at about 6.
When the solution of the nitrogen containing heterocyclic compound to be treated, or the incubation mixture, is at an acid pH outside the permissible range, the pH can be adjusted to a value that permits the conversion of the nitrogen containing heterocyclic compound to be treated using a base, this base being either in solution or a solid. Preferably, the base is a solid, so that the solution of nitrogen containing eterocyclic compound to be treated is not diluted. It can, for example, advantageously be in the form of pellets. This base can be chosen, for example, from within the group comprising NaOH and KOH.
According to the method of the invention, bringing the micro-organism into contact with the solution of the nitrogen containing heterocyclic compound to be treated is carried out at a temperature, preferably a substantially constant temperature that permits the culture of the micro-organism in an incubation mixture and the conversion of the nitro groups, that is to say, a temperature that is favorable to a culture of the micro-organism and to said conversion. When the micro-organism is a bacterium, in general, this temperature is between 20 and 35xc2x0 C., preferably from 20 to 30xc2x0 C. When the bacterium is Bacillus licheniformis, this temperature is preferably within the range 20 to 30xc2x0 C. and more preferably at about 25xc2x0 C.
The bringing into contact can be carried out for example in a fermenter, which can be a means of keeping the temperature substantially constant during the conversion of the nitro groups into amine groups.
In addition, the fermenter can be fitted with a stirrer to maintain agitation during the bringing into contact.
According to the method of the invention, an agent for inducing the general reducing metabolism of the micro-organism used can be added to the solution of the nitrogen containing heterocyclic compound comprising at least one nitro group, or to the incubation mixture, that brings it into contact with the micro-organism. This inducing agent is added to the solution or to the mixture to increase the general reducing metabolism of the micro-organism thereby encouraging the conversion of the nitro group or groups of the nitrogen containing heterocyclic compound into amine groups.
An optimum concentration of inducing agent for the general reducing metabolism can be determined by carrying out conversion tests on a nitrogen containing heterocyclic compound comprising a nitro group as a function of the concentration of inducing agent in an incubation mixture.
This inducing agent may be any compound that is able to induce the general reducing metabolism of the micro-organism used, for example, glucose, glucose polymers, molasses or maize hydrosylate etc.
When the agent for inducing the general reducing metabolism of the chosen micro-organism is glucose, its concentration in the solution can be, for example, about 0 to 3.0 g per 100 ml solution, preferably about 1 to 2 g per 100 ml of solution.
For example, for one liter of a solution of the nitrogen containing heterocyclic compound ONTA at 15 g/l, a biomass of Bacillus licheniformis of 90 g, a quantity of sugar of 15 g/l, a pH of about 6 and a temperature of 25xc2x0 C., the method of the invention permits greater than 80% conversion of ONTA into the corresponding amine, and even all of it in 24 hours.
The method conforming to the invention therefore allows one to treat an aqueous industrial effluent comprising a toxic nitrogen containing heterocyclic compound to be treated, such as, for example ONTA. When the nitrogen containing heterocyclic compound to be treated has been converted by the micro-organism in the effluent to be treated into the corresponding amine, this treated effluent can, for example, be treated to remove the amine formed, by for example, gel filtration, for example on Sephadex G10.
The invention also relates to a method for the synthesis of 5-amino-1,2,4-triazole-3-one that comprises bringing a solution of 5-nitro-1,2,4-triazole-3-one into contact with a micro-organism capable of reducing the 5-nitro group into a 5-amino group.
The solution of 5-amino-1,2,4-triazole-3-one can be an aqueous solution of this compound, prepared at a concentration that can range up to the saturation concentration.
This method of synthesis can be carried out in the same manner as the method of converting a nitro group in a nitrogen containing heterocyclic compound to be treated according to the invention.
According to the method of the invention, the micro-organism can be selected by screening of micro-organisms that are living or are able to live in an industrial processing effluent arising from the manufacture of explosives, in foul water, and in a general way, in a medium, called in this description, a source medium, preferably comprising nitrogen containing heterocyclic compounds preferably comprising at least one nitro group.
For example, the screening can be carried out starting with a sample of a medium that can be treated according to the method of the invention.
According to the method of the invention, the micro-organism can be of the Bacillus licheniformis type.
According to the method of the invention, the Bacillus licheniformis is the Bacillus licheniformis colony LCM2 filed under number I-1915 in the CNCM held by the Pasteur Institute in France.
According to the invention, when the synthesis of 5-amino-1,2,4-triazole-3-one has been completed, it can be isolated from the mixture of solution and biomass by centrifugation to remove the biomass and by purification of the 5-amino-1,2,4-triazole-3-one contained in the supernatant liquid from the centrifugation.
The treatment can, for example, be carried out by evaporation of the aqueous phase and purification of the 5-amino-1,2,4-triazole-3-one extract by traditional purification methods.
The invention also relates to a colony of the Bacillus licheniformis type filed under the number I-1915 in the CNCM (Collection Nationale de Culture de Micro-organisms) held by the Pasteur Institute in France.